This proposal is focused on basic cell biological mechanisms of a poorly understood membrane trafficking pathway- the endosome-to-Golgi "bypass" pathway- in three human disease-related contexts. The first is cell invasion by bacterial toxins, which co-opt the bypass pathway causing human disease. The second is manganese toxicity, which induces downregulation of a key component of the bypass pathway and neurodegenerative disorders in humans. The third is the generation of a promising early onset serum marker for liver cancer due to alterations in bypass pathway trafficking in hepatocellular carcinoma. Protein and lipid cargo originating at the cell surface or Golgi apparatus gain access to the pathway by moving to early/sorting endosomes. The cargo then "bypasses" the degradative branch of the endocytic pathway involving late endosomes and instead traffics directly to the Golgi apparatus. We are one of the few labs elucidating the mechanisms that govern trafficking in this pathway. Our approach is to identify proteins trafficking in the bypass pathway, map their targeting signals and interactions, and test their functional role using biochemical and morphological assays in conjunction with siRNA-mediated knockdown and gene replacement. Our published (Bachert et al., 2001;Linstedt et al., 1997;Natarajan and Linstedt, 2004;Puri et al., 2002;Puthenveedu et al., 2003) and unpublished work indicates that a) Golgi proteins GPP130 and GP73 cycle in the bypass pathway and their pH-sensitive Golgi targeting depends on this cycling, b) GPP130 and GP73 bypass pathway cycling involves the combined use of Golgi and endosomal determinants present in their lumenal coiled-coil domains, c) GPP130 shows pH-sensitive binding to a large lumenal complex, d) analogous lumenal determinant-mediated sorting of GPP130 occurs in polarized cells and is basolaterally restricted, e) GPP130 is selectively required for bypass pathway trafficking of Shiga toxin and other bypass markers and this functioning depends on both the GPP130 lumenal sorting determinants and the cytoplasmic domain, f) the COG docking complex is also required and acts downstream of GPP130, g) in response to low concentrations of manganese GPP130 trafficking is rapidly and selectively altered leading to GPP130 downregulation, and h) GP73 trafficking in the bypass pathway is altered in HCC such that it is cleaved and its ectodomain is released from cells producing a promising biomarker of the disease. Thus, our major aim is to test the hypothesis that GPP130 and similarly structured bypass pathway components use lumenal, pH-sensitive interactions to be incorporated into a large Golgi retrieval complex that buds from sorting and recycling endosomes. By interacting with this complex, Shiga-like toxins access the bypass pathway. During its formation, the retrieval complex also interacts with transport components essential for efficient bypass pathway trafficking- including the COG docking complex- and the GPP130 cytoplasmic domain plays a critical role in this recruitment. In addition, we will determine the mechanism by which Mn+2 exposure and cellular transformation alter bypass trafficking causing GPP130 downregulation and GP73 ectodomain shedding, respectively. PUBLIC HEALTH RELEVANCE: Defects in membrane trafficking are responsible for many human diseases and our understanding of the molecular basis of these defects is paving the way to future effective therapeutics. An important, but understudied, example is the bypass membrane trafficking pathway from the cell surface to its interior compartments. Our investigation into the mechanism of bypass pathway trafficking is revealing novel and potentially clinically relevant insights into retrieval-based targeting of Golgi proteins, infection of humans by Shiga-like toxins, manganese neurotoxicity (a major problem due to contaminated well water in underdeveloped nations), and production of an early serum biomarker of human liver cancer (the fifth-leading cause of cancer death worldwide). Thus, the work offers possibilities for new modes for drug delivery to the cytosolic compartment, treatment of infectious diseases where bacterial toxins are involved, therapies against manganese-induced motor dysfunction syndrome, and non-invasive early detection of liver cancer.